Project Summary. The long-term goal of this project is to understand how transcription by RNA polymerase II (RNApII) is coupled to RNA processing and termination. This project previously developed a model in which the C-terminal domain (CTD) of the RNApII subunit Rpb1 displays characteristic phosphorylation patterns at different stages of the transcription cycle to promote binding of the appropriate factors for co-transcriptional RNA processing. The fundamental knowledge generated by this project provides significant insight into how the CTD phosphorylation cycle affects medically important processes such as the stimulation of HIV transcription by the viral Tat protein and pausing of RNApII at developmentally regulated genes. This project is necessary to better understand both the enzymes that mediate the changes in CTD phosphorylation (kinases, phosphatases, etc.) as well as the proteins that recognize these patterns. In the next funding period, three specific aims will be pursued, with a focus on measuring dynamics of events during transcription. The first aim continues our work directly analyzing CTD phosphorylation sites by mass spectrometry, avoiding pitfalls associated with the monoclonal antibodies used in most CTD studies. A modified CTD (msCTD) was engineered to discriminate individual proximal and distal repeats by mass. Recent improvements to peptide chromatography and computational analysis will improve our accuracy and throughput. In vivo CTD phosphorylations will be analyzed in cells where various CTD modifying enzymes are rapidly inactivated or depleted. Analysis of RNApII associated with specific CTD binding proteins or CTD antibodies will also be performed to determine their binding specificities. The second aim exploits our recent discovery that CTD cycle progresses as a function of time, rather than elongation distance. This realization allowed us to create an in vitro system that reproduces the progression of CTD phosphorylations and associated factors on elongation complexes, facilitating real time analysis of dynamics. This immobilized template system will be combined with the msCTD from Aim 1 to produce a high-resolution time course of CTD phosphorylation, probing the contributions of individual kinases and phosphatases. The third aim adapts the immobilized template assay to single-molecule microscopy. We can visualize individual transcription events with up to three fluorescently-labeled transcription factors, providing second to millisecond time resolution of binding kinetics. We will measure the stoichiometries, order of binding, and cooperative interactions between multiple CTD binding and elongation complex factors. Altogether, this project will make a unique contribution to our understanding of gene expression by providing a time-resolved picture of events that complements inherently static techniques such as structural studies or genomics.